Multifunctional Nanoparticle System Research Articles

Synthesis and Characterization of AICAR and DOX Conjugated Multifunctional Nanoparticles as a Platform for Synergistic Inhibition of Cancer Cell Growth.

The success of cancer treatment depends on the response to chemotherapeutic agents. However, malignancies often acquire resistance to drugs if they are used frequently. Combination therapy involving both a chemotherapeutic agent and molecularly targeted therapy may have the ability to retain and enhance therapeutic efficacy. Here, we addressed this issue by examining the efficacy of a novel therapeutic strategy that combines AICAR and DOX within a multifunctional platform. In this context, we reported the bottom-up synthesis of Fe3O4@SiO2(FITC)-FA/AICAR/DOX multifunctional nanoparticles aiming to neutralize survivin (BIRC5) to potentiate the efficacy of DOX against chemoresistance. The structure of nanoparticles was characterized by dynamic light scattering (DLS), zeta-potential measurement, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), and electron microscopy (SEM and STEM with EDX) techniques. Cellular uptake and cytotoxicity experiments demonstrated preferentially targeted delivery of nanoparticles and an efficient reduction of cancer cell viability in five different tumor-derived cell lines (A549, HCT-116, HeLa, Jurkat, and MIA PaCa-2). These results indicate that the multifunctional nanoparticle system possesses high inhibitory drug association and sustained cytotoxic effect with good biocompatibility. This novel approach which combines AICAR and DOX within a single platform might be promising as an antitumor treatment for cancer.

Combination Nanopreparations of a Novel Proapoptotic Drug - NCL-240, TRAIL and siRNA.

To develop a multifunctional nanoparticle system carrying a combination of pro-apoptotic drug, NCL-240, TRAIL [tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand] and anti-survivin siRNA and to test the combination preparation for anti-cancer effects in different cancer cells. Polyethylene glycol-phosphoethanolamine (PEG-PE) - based polymeric micelles were prepared carrying NCL-240. These micelles were used in combination with TRAIL-conjugated micelles and anti-survivin siRNA-S-S-PE containing micelles. All the micelles were characterized for size, zeta potential, and drug encapsulation efficiency. Different cancer cells were used to study the cytotoxicity potential of the individual as well as the combination formulations. Other cell based assays included cellular association studies of transferrin-targeted NCL-240 micelles and study of cellular survivin protein downregulation by anti-survivin siRNA-S-S-PE containing micelles. NCL-240 micelles and the combination NCL-240/TRAIL micelles significantly increased cytotoxicity in the resistant strains of SKOV-3, MCF-7 and A549 as compared to free drugs or single drug formulations. The NCL-240/TRAIL micelles were also more effective in NCI/ADR-RES cancer cell spheroids. Anti-survivin siRNA micelles alone displayed a dose-dependent reduction in survivin protein levels in A2780 cells. Treatment with NCL-240/TRAIL after pre-incubation with anti-survivin siRNA inhibited cancer cell proliferation. Additionally, a single multifunctional system composed of NCL-240/TRAIL/siRNA PM also had significant cytotoxic effects in vitro in multiple cell lines. These results demonstrate the efficacy of a combination of small-molecule PI3K inhibitors, TRAIL, and siRNA delivered by micellar preparations in multiple cancer cell lines.

Amifostine-conjugated pH-sensitive calcium phosphate-covered magnetic-amphiphilic gelatin nanoparticles for controlled intracellular dual drug release for dual-targeting in HER-2-overexpressing breast cancer

We developed a surfactant-free method utilizing amifostine to stably link a targeting ligand (Herceptin) to amphiphilic gelatin (AG)-iron oxide@calcium phosphate (CaP) nanoparticles with hydrophobic curcumin (CUR) and hydrophilic doxorubicin (DOX) encapsulated in the AG core and CaP shell (AGIO@CaP-CD), respectively. This multi-functional nanoparticle system has a pH-sensitive CaP shell and degradable amphiphilic gelatin (AG) core, which enables controllable sequential release of the two drugs. The dual-targeting system of AGIO@CaP-CD (HER-AGIO@CaP-CD) with a bioligand and magnetic targeting resulted in significantly elevated cellular uptake in HER2-overexpressing SKBr3 cells and more efficacious therapy than delivery of targeting ligand alone due to the synergistic cell multi-drug resistance/apoptosis-inducing effect of the CUR and DOX combination. This nanoparticle combined with Herceptin and iron oxide nanoparticles not only provided a dual-targeting functionality, but also encapsulated CUR and DOX as a dual-drug delivery system for the combination therapy. This study further demonstrated that the therapeutic efficacy of this dual-targeting co-delivery system can be improved by modifying the application duration of magnetic targeting, which makes this combination therapy system a powerful new tool for in vitro/in vivo cancer therapy, especially for HER2-positive cancers.

Tumor-targeted and pH-controlled delivery of doxorubicin using gold nanorods for lung cancer therapy.

BackgroundIn lung cancer, the efficacy of conventional chemotherapy is limited due to poor drug accumulation in tumors and nonspecific cytotoxicity. Resolving these issues will increase therapeutic efficacy.MethodsGNR-Dox-Tf-NPs (gold nanorod-doxorubicin-transferrin-nanoparticles) were prepared by different chemical approaches. The efficacy of these nanoparticles was carried out by cell viability in lung cancer and primary coronary artery smooth muscle cells. The receptor-mediated endocytosis studies were done with human transferrin and desferrioxamine preincubation. The GNR-Dox-Tf nanoparticles induced apoptosis, and DNA damage studies were done by Western blot, H2AX foci, and comet assay.ResultsWe developed and tested a gold nanorod-based multifunctional nanoparticle system (GNR-Dox-Tf-NP) that carries Dox conjugated to a pH-sensitive linker and is targeted to the transferrin receptor overexpressed in human lung cancer (A549, HCC827) cells. GNR-Dox-Tf-NP underwent physicochemical characterization, specificity assays, tumor uptake studies, and hyperspectral imaging. Biological studies demonstrated that transferrin receptor-mediated uptake of the GNR-Dox-Tf-NP by A549 and HCC827 cells produced increased DNA damage, apoptosis, and cell killing compared with nontargeted GNR-Dox-NP. GNR-Dox-Tf-NP-mediated cytotoxicity was greater (48% A549, 46% HCC827) than GNR-Dox-NP-mediated cytotoxicity (36% A549, 39% HCC827). Further, GNR-Dox-Tf-NP markedly reduced cytotoxicity in normal human coronary artery smooth muscle cells compared with free Dox.ConclusionThus, GNR-Dox-Tf nanoparticles can selectively target and deliver Dox to lung tumor cells and alleviate free Dox-mediated toxicity to normal cells.

Chitosan coated polylactic acid nanoparticle-mediated combinatorial delivery of cisplatin and siRNA/Plasmid DNA chemosensitizes cisplatin-resistant human ovarian cancer cells.

Development of resistance toward anticancer drugs results in ineffective therapy leading to increased mortality. Therefore, overriding resistance and restoring sensitivity to anticancer drugs will improve treatment efficacy and reduce mortality. While numerous mechanisms for drug resistance in cancer have previously been demonstrated, recent studies implicate a role for proteasome and the autophagy regulatory protein P62/SQSTM1 (P62) in contributing to drug resistance. Specifically, reduction in the expression of the β5 subunit of the proteasome and/or enhanced P62 protein expression is known to contribute to cancer drug resistance such as cisplatin (CDDP) in ovarian cancer cells. Therefore, we hypothesized that restoration of β5 expression and/or suppression of P62 protein expression in CDDP-resistant ovarian cancer cells will lead to restoration of sensitivity to CDDP and enhanced cell killing. To test our hypothesis we developed a biodegradable multifunctional nanoparticle (MNP) system that codelivered P62siRNA, β5 plasmid DNA, and CDDP and tested its efficacy in CDDP resistant 2008/C13 ovarian cancer cells. MNP consisted of CDDP loaded polylactic acid nanoparticle as inner core and cationic chitosan (CS) consisting of ionically linked P62siRNA (siP62) and/or β5 expressing plasmid DNA (pβ5) as the outer layer. The MNPs were spherical in shape with a hydrodynamic diameter in the range of 280-350 nm, and demonstrated encapsulation efficiencies of 82% and 78.5% for CDDP and siRNA respectively. MNPs efficiently protected the siRNA and showed superior serum stability compared to naked siRNA as measured by gel retardation and spectrophotometry assays. The MNPs successfully delivered siP62 and pβ5 to cause P62 knockdown and restoration of β5 expression in 2008/C13 cells. Combined delivery of siP62, pβ5, and CDDP using the MNPs resulted in a marked reduction in the IC50 value of CDDP in 2008/C13 cells from 125 ± 1.3 μM to 98 ± 0.6 μM (P < 0.05; 21.6% reduction) when compared to the reduction in the IC50 of CDDP observed in cells that had only siP62 delivered (IC50 = 106 ± 1.1 μM; P < 0.05; 15.2% reduction) or pβ5 delivered (IC50 = 115 ± 2.8 μM; 8% reduction) via MNPs. Finally, our studies showed that the CDDP resistance index in 2008/C13 cells was reduced from 4.62 for free CDDP to 3.62 for MNP treatment. In conclusion our study results demonstrated the efficacy of our MNP in overcoming CDDP resistance in ovarian cancer cells.

Quantum-Dot-Conjugated Graphene as a Probe for Simultaneous Cancer-Targeted Fluorescent Imaging, Tracking, and Monitoring Drug Delivery

We report a novel quantum-dot-conjugated graphene, i.e., hybrid SiO2-coated quantum dots (HQDs)-conjugated graphene, for targeted cancer fluorescent imaging, tracking, and monitoring drug delivery, as well as cancer therapy. The hybrid SiO2 shells on the surface of QDs not only mitigate its toxicity, but also protect its fluorescence from being quenched by graphene. By functionalizing the surface of HQDs-conjugated graphene (graphene-HQDs) with transferrin (Trf), we developed a targeted imaging system capable of differential uptake and imaging of cancer cells that express the Trf receptor. The widely used fluorescent antineoplastic anthracycline drug, doxorubicin (DOX), is adsorbed on the surface of graphene and results in a large loading capacity of 1.4 mg mg(-1). It is advantageous that the new delivery system exhibits different fluorescence color in between graphene-HQDs and DOX in the aqueous core upon excitation at a same wavelength for the purpose of tracking and monitoring drug delivery. This simple multifunctional nanoparticle system can deliver DOX to the targeted cancer cells and enable us to localize the graphene-HQDs and monitor intracellular DOX release. The specificity and safety of the nanoparticle conjugate for cancer imaging, monitoring, and therapy has been demonstrated in vitro.

Enhanced cell uptake of superparamagnetic iron oxide nanoparticles through direct chemisorption of FITC-Tat-PEG600-b-poly(glycerol monoacrylate)

Magnetic nanoparticles (MNPs) functionalized with specific ligands are emerging as a highly integrated platform for cancer targeting, drug delivery, and magnetic resonance imaging applications. In this study, we describe a multifunctional magnetic nanoparticle system (FITC-Tat MNPs) consisting of a fluorescently labeled cell penetrating peptide (FITC-Tat peptide), a biocompatible block copolymer PEG600-b-poly(glycerol monoacrylate) (PEG600-b-PGA), and a superparamagnetic iron oxide (SPIO) nanoparticle core. The particles were prepared by direct chemisorption of PEG600-b-PGA conjugated with FITC-Tat peptide on the SPIO nanoparticles. FITC-MNPs without Tat were prepared for comparison. Flow cytometry assays revealed significantly higher uptake of FITC-Tat MNPs compared to FITC-MNPs in Caco-2 cells. These results were confirmed using confocal laser scanning microscopy (LSCM), which further demonstrated that the FITC-Tat MNPs accumulated in the cytoplasm and nucleus while the FITC-MNPs were localized in the cell membrane compartments. The FITC-Tat MNPs did not exhibit observable cytotoxicity in MTS assays.

Multifunctional silica nanoparticles for targeted delivery of hydrophobic imaging and therapeutic agents

This article reports the development of a multifunctional silica nanoparticle system for targeted delivery of hydrophobic imaging and therapeutic agents. Normally, silica nanoparticles have been widely used to deliver hydrophilic drugs such as doxorubicin while difficult to carry hydrophobic drugs. A strategy for loading hydrophobic drugs onto silica nanoparticles via covalent attachment was developed in this study as a universal strategy to solve this problem. Docetaxel, one of the most potent therapeutics for cancer treatment is selected as a model hydrophobic drug and quantum dots (QDs) are used as a model imaging agent. Such a multifunctional delivery system possesses high drug loading capacity, controlled drug release behavior and stable drug reservation. A mixed layer of polyethylene glycol conjugated phospholipids is formed on the nanoparticle surface to further enhance the biocompatibility and cell fusion capability of the delivery system. Folic acid as ligand is then conjugated onto the surface layer for targeting. Such a multifunctional system for targeting, imaging and therapy is characterized and evaluated in vitro. Fluorescent confocal microscopy is used to monitor the cellular uptake by specific cancer cells. Cytotoxicity studies are conducted by using MTT assay.

Targeted dual-contrast T1- and T2-weighted magnetic resonance imaging of tumors using multifunctional gadolinium-labeled superparamagnetic iron oxide nanoparticles

Development of a multifunctional nanoparticle (NP) system allowing for dual-contrast T 1- and T 2-weighted targeted magnetic resonance (MR) imaging of tumors could significantly improve the diagnosis accuracy. In this study, superparamagnetic silica-coated iron oxide core-shell nanoparticles (Fe 3O 4@SiO 2 NPs) with a diameter of approximately 21 nm were synthesized via a thermal decomposition approach and were aminated through silanization. The amine-functionalized Fe 3O 4@SiO 2 NPs enabled the covalent conjugation of a paramagnetic gadolinium complex (Gd-DTPA, DTPA: diethylenetriamine pentaacetic acid) and an arginine-glycine-aspartic acid (RGD) peptide as a targeting ligand onto their surface. The formed Fe 3O 4@SiO 2(Gd-DTPA)-RGD NPs are water-dispersible, stable, and biocompatible as confirmed by MTT cell viability assay. Relaxivity measurements show that they have a T 1 relaxivity ( r 1) of 4.2 m m −1 s −1 and T 2 relaxivity ( r 2) of 17.4 m m −1 s −1 at the Gd/Fe molar ratio of 0.3:1, suggesting a possibility to use them as both T 1 positive and T 2 negative contrast agents. In vitro and in vivo MR imaging experiments show that the developed multifunctional Fe 3O 4@SiO 2(Gd-DTPA)-RGD NPs enable targeted dual-contrast T 1- and T 2-weighted MR imaging of tumor cells over-expressing high-affinity α vβ 3 integrin in vitro and in vivo. Our results clearly indicate that the approach to forming multifunctional Fe 3O 4@SiO 2(Gd-DTPA)-RGD NPs could be extended for fabricating other biologically active NPs for T 1- and T 2-weighted MR imaging of other biological systems with high accuracy.

Conjugated Polymer Nanoparticles for Drug Delivery and Imaging

We prepared a new conjugated polymer nanoparticle with the size of about 50 nm that is prepared by electrostatic assembly of cationic conjugated polymer PFO and anionic poly(l-glutamic acid) conjugated with anticancer drug doxorubicin (PFO/PG-Dox). The PFO exhibits good fluorescence quantum yield, photostability, and little cytotoxicity to meet the essential requests for cell imaging. In PFO/PG-Dox nanoparticles, the fluorescence of PFO is highly quenched by Dox by electron transfer mechanism, and thus the PFO is in the fluorescence "turn-off' state. After PFO/PG-Dox nanoparticles are exposed to carboxypeptidase or are taken up by cancer cells, the poly(l-glutamic acid) is hydrolysed to release the Dox, inducing the activation of PFO fluorescence to "turn-on" state. This multifunctional nanoparticle system can deliver Dox to targeted cancer cells and monitor the Dox release based on fluorescence "turn-on" signal of PFO, which concurrently images the cancer cells. The present work opens the door for new functional studies of conjugated polymer in simultaneous imaging and disease therapeutics.

Multifunctional Nanoparticles of Biodegradable Copolymer Blend for Cancer Diagnosis and Treatment

A multifunctional nanoparticle (NP) system is developed to provide a sustained, controlled and targeted co-delivery of quantum dots (QDs) as a model imaging agent and docetaxel as a model anticancer drug. The NPs are made of a polymeric blend of poly(lactic-co-glycolic acid), which forms a biodegradable NP matrix, and the novel copolymer D-alpha-tocopheryl polyethylene glycol 1000 succinate-COOH, which facilitates ligand conjugation on the NP surface. The NPs were prepared by nanoprecipitation and characterized for their size and size distribution, surface morphology, surface charge, QD/drug encapsulation and loading efficiency, and in vitro drug release profile. The targeting effects of such NPs were evaluated both quantitatively and qualitatively through the cellular uptake of the QDs as well as the cytotoxicity of the drug using MCF-7 cells, which overexpress folate receptors and NIH 3T3 cells, which have no folate receptors overexpression. NPs with folate conjugated on their surface achieved much higher cellular uptake than those with no folate conjugation in MCF-7 cells while no significant targeting effect could be observed for NIH 3T3 cells. The drug formulated in the folate-conjugated NPs were more efficious compared with NPs with no folate conjugation as well as the current clinical formulation Taxotere.

Design of a multifunctional heparin-based nanoparticle system for anticancer drug delivery

A ligand-mediated nanoparticulate drug carrier composed of heparin-based copolymers was designed to achieve targeted drug delivery for chemotherapy. Novel amphiphilic copolymers composed of heparin with antitumor activities and poly(β-benzyl-L-aspartate) (PBLA) with the potential capacity for serving as pH-responsive drug release reservoirs were synthesized by conjugating PBLA as a hydrophobic segment to the hydrophilic heparin backbone. To introduce folate ligands for tumor targeting to the heparin/PBLA copolymers, folate-conjugated heparin/PBLA copolymers were prepared with various compositions by varying the feed molar ratio of PBLA, amineterminated folate (or folate-conjugated PEG) to heparin. The synthesis and composition of the heparin-based copolymers were confirmed by 1H NMR and colorimetric methods, respectively. Heparin-based amphiphilic copolymeric nanoparticles were prepared by micelle formation in an aqueous solution. The properties of the micelles were described by the critical micelle concentration (CMC), size and size distribution. The CMC values of heparin-based copolymers decreased with increasing concentration of the PBLA hydrophobic segments in the copolymers. The conjugation of folate (or folate-conjugated PEG) with hydrophilic nature into heparin/PBLA copolymers affected the free chain-micelle equilibrium, leading to an increase in the CMC values. The particle size of the heparin-based copolymers determined by light scattering measurements ranged from 100 to 130 nm, depending on the molecular weight and composition of the copolymers. Field emission scanning electron microscopy showed that the heparinbased copolymeric particles had a sub-micron size with spherical shape.

Multifunctional core-shell polymeric nanoparticles for transdermal DNA delivery and epidermal Langerhans cells tracking

Skin is a highly immune-reactive tissue containing abundant antigen-presenting cells such as Langerhans cells (LCs), and thus is a favorable site for DNA immunization. This study developed a multifunctional core-shell nanoparticle system, which can be delivered transdermally into the epidermis via a gene gun, for use as a DNA carrier. The developed nanoparticles comprised a hydrophobic PLGA core and a positively-charged glycol chitosan (GC) shell. The core of the nanoparticles was used to load fluorescent quantum dots (QDs) for ultrasensitive detection of Langerhans cell migration following transdermal delivery, while a reporter gene was electrostatically adsorbed onto the GC shell layer of the nanoparticles. Results of fluorescence spectrophotometry, transmission electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction measurement confirmed that the prepared nanoparticles had a core-shell structure with QDs in their core area. The surface charge of nanoparticles depended strongly on pH environment, enabling the intracellular release of the loaded DNA via a pH-mediated mechanism. Using a mouse model, this study demonstrated that bombardment of nanoparticles transfected DNA directly into LCs present in the epidermis; the transfected LCs then migrated and expressed the encoded gene products in the skin draining lymph nodes. These observation results suggest that the developed nanoparticle system is suitable for monitoring and fine-tuning important functional aspects of the immune system, in conjunction with the loaded fluorescence, and thus has potential for use in immunotherapy and vaccine development.

Tumor-Targeted Drug Delivery and MRI Contrast Enhancement by Chlorotoxin-Conjugated Iron Oxide Nanoparticles

This study examines the capabilities of an actively targeting superparamagnetic nanoparticle to specifically deliver therapeutic and MRI contrast agents to cancer cells. Iron oxide nanoparticles were synthesized and conjugated to both a chemotherapeutic agent, methotrexate, and a targeting ligand, chlorotoxin, through a poly(ethylene glycol) linker. Cytotoxicity of this nanoparticle conjugate was evaluated by Alamar Blue cell viability assays, while tumor-cell specificity was examined in vitro and in vivo by MRI. Characterization of these multifunctional nanoparticles confirms the successful attachment of both drug and targeting ligands. The targeting nanoparticle demonstrated preferential accumulation and increased cytotoxicity in tumor cells. Furthermore, prolonged retention of these nanoparticles was observed within tumors in vivo. The improved specificity, extended particle retention and increased cytotoxicity toward tumor cells demonstrated by this multifunctional nanoparticle system suggest that it possesses potential for applications in cancer diagnosis and treatment.

Quantum Dot−Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer

We report a novel quantum dot (QD)-aptamer(Apt)-doxorubicin (Dox) conjugate [QD-Apt(Dox)] as a targeted cancer imaging, therapy, and sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD-Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD-Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor-acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor-quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.

Ultrafast Energy Migration in Chromophore Shell−Metal Nanoparticle Assemblies

A multifunctional ligand-coated nanoparticle system containing approximately 2000 highly two-photon absorptive chromophores has been investigated by means of steady-state and femtosecond time-resolved spectroscopy. This system with a high local concentration of chromophores showed remarkably low self-quenching and a high fluorescence quantum yield, which is important for a variety of two-photon sensing and imaging applications. We have observed evidence for ultrafast energy migration in these chromophore shell-metal nanoparticle systems. Time-resolved experiments also showed non-zero residual anisotropy after the initial fast decay, which can be interpreted as due to the formation of the specific domains on the metal surfaces. This investigation opens new avenues toward the development of multi-chromophoric efficient TPA fluorescence sensing/imaging systems with large numbers of chromophores per one metal particle nanoparticle.

Co nanoparticle monolayer prepared by multiple diffusive incorporations onto a pre-existing nanoparticle template

A monolayer of Co nanoparticles was fabricated on a polyimide (PI) film by successively depositing a 3.5-nm-thick Co thin film on top of a pre-existing nano-sized Ni particle template. Each Co deposition was followed by thermal annealing to induce coalescence of the Co film onto the pre-existing Ni seed particles. The Co deposition/thermal annealing step was repeated up to three times to grow 7–10-nm-sized Co nanoparticles on a polyimide film. We demonstrate that the average particle size can be controlled by the number of Co depositions. Thus-produced Co nanoparticles exhibited saturation magnetization comparable to its bulk value with coercivity reaching up to 120 Oe. Such a nanoparticle template process presented here can be easily extended to a ternary alloy particle and multi-functional nanoparticle system for potential applications in nanomagnet arrays, nanophotonic arrays or nanocatalyst arrays.